*Background/Disease Information (sort of like the Intro to your Mini Research Write up): Tuberculosis is caused by the Mycobacterium tuberculosis. It is an airborne disease that mainly attacks the respiratory system. Most cases of tuberculosis are latent and do no exhibit any symptoms. However, about 10% of latent cases will develop into active tuberculosis. Roughly 50% of cases of active tuberculosis are fatal. Roughly one-third of the global population have had tuberculosis. Infection is primarily in developing countries. Over 1 million people die from tuberculosis each year. Tuberculosis is diagnosed by a tuberculosis skin test, but it is difficult to treat the disease. Symptoms of active TB include a coughing that last longer than 3 weeks, chest pain, coughing up blood, weakness, weight less, loss of appetite, chills, fever, night sweats.
Link to TDR Targets page (if present): None present
Essentiality of this protein: "myo-inositol is a key metabolite for mycobacteria" (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1222319/) "Phosphatidylinositol is an essential component of mycobacteria, and phosphatidylinositolbased lipids such as phosphatidylinositolmannosides, lipomannan, and lipoarabinomannan are major immunomodulatory components of the Mycobacterium tuberculosis cell wall. Inositol monophosphatase (EC 3.1.3.25) is a crucial enzyme in the biosynthesis of free myo-inositol from inositol-1-phosphate, a key substrate for the phosphatidylinositol synthase in mycobacteria." (http://pubs.acs.org/doi/pdf/10.1021/bi0160056)
"IMP activity was determined by quantifying the inorganic phosphate released from substrate using the Malachite Green procedure modified according to Baykov et al. (19). Standard assays were conducted in 100-µL reaction mixture aliquots including 50 mM Tris-HCl pH 8.5, 0.4 mM I-1-P, 10 mM Mg2+ and 1 µg of the purified protein. The mixture was incubated at 37 °C for 1 min. The reaction was stopped by the addition of 700 µL of water and 200 µL of the malachite green reagent. The reagent contains 2.4 M H2SO4, which is sufficient to stop the reaction. The release of inorganic phosphate (Pi) was determined by measuring the OD at 630 nm in comparison to that of standard Pi samples. Control assays included substrate and Mg2+ but no enzyme or enzyme alone. For the determination of the Mg2+-dependent enzyme profile, pH optimum, Km, metal ion dependence, monovalent cation inhibition, substrate specificity, and temperature dependence, the corresponding factors were varied. For the determination of thermal stability, the enzyme was incubated for different periods of time at 70 °C in the presence or absence of 10 mM MgCl2. The activity was then measured by incubating the enzyme for 1 min at 37 °C in the presence of the substrate and 10 mM MgCl2." (http://pubs.acs.org/doi/pdf/10.1021/bi0160056)
Expression Information (has it been expressed in bacterial cells): E. Coli C41 (DE3) pET23b-suhB plasmid Method: "An overnight culture of E. coli C41 (DE3) pET23b-suhB was used to inoculate a large volume of LB broth supplemented with 100 µg/mL ampicillin (Sigma) and incubated at 37 °C under shaking until the optical density (OD) at 600 nm reached 0.5. The culture was then transferred to fresh terrific broth supplemented with 100 µg/mL ampicillin and induced with 0.1 mM isopropyl-â-D-thiogalactopyranoside (IPTG) (Promega) overnight at 16 °C." (http://pubs.acs.org/doi/pdf/10.1021/bi0160056)
Purification Method: Nickel column "The cells [E. Coli] were harvested by centrifugation and resuspended in 50 mM MOPS buffer pH 7.9. Bacteria were disrupted by probe sonication (Soniprep 150, MSE Sanyo Gallenkamp, Crawley, Sussex, U.K.; 1-cm probe) for a total time of 2.5 min in 30-s pulses with 45-s cooling intervals between pulses. The resulting extracts were centrifuged at 27000g for 20 min at 4 °C. The supernatant was collected and applied to a Ni2+-charged His-trap column (1 mL, Amersham Pharmacia Biotech) that had been equilibrated with 125 mM phosphate buffer (pH 7.5) and 0.5 M NaCl. The column was extensively washed with 50 mM imidazole in the above buffered solution and eluted with a stepwise gradient of imidazole (50-500 mM). One milliliter fractions were collected, and the presence and purity of SuhB was detected by 15% SDS-PAGE. Fractions containing pure SuhB were dialyzed against 50 mM Tris/ HCl pH 7.5 and stored at -20 °C. Protein concentrations were determined using the BCA protein assay kit (Pierce; Rockford, IL)." (http://pubs.acs.org/doi/pdf/10.1021/bi0160056)
Image of protein (PyMol with features delineated and shown separately):
Figure 2. Pymol render of Inositol Monophosphatase of Mycobacterium tuberculosis.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MTRPDNEPARLRSVAENLAAEAAAFVRGRRAEVFGISRAGDGDGAVRAKSSPTDPVTVVD TDTERLLRDRLAQLRPGDPILGEEGGGPADVTATPSDRVTWVLDPIDGTVNFVYGIPAYA VSIGAQVGGITVAGAVADVAARTVYSAATGLGAHLTDERGRHVLRCTGVDELSMALLGTG FGYSVRCREKQAELLAHVVPLVRDVRRIGSAALDLCMVAAGRLDAYYEHGVQVWDCAAGA LIAAEAGARVLLSTPRAGGAGLVVVAAAPGIADELLAALQRFNGLEPIPD
*length of your protein in Amino Acids: 290 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 30,027 kD
Molar Extinction coefficient of your protein at 280 nm wavelength: 20190 M-1 cm-1
Figure 3. TMPred graph of Inositol Monophosphatase of Mycobacterium tuberculosis run using amino acid sequence.
*CDS Gene Sequence (paste as text only): GTGACACGACCTGACAACGAACCCGCGCGGCTGCGCTCTGTGGCCGAAAACCTTGCTGCCGAGGCGGCGG CCTTCGTTCGCGGTCGTCGGGCCGAGGTTTTCGGCATCTCCAGGGCGGGCGACGGCGACGGCGCGGTGCG CGCGAAGAGCAGCCCGACCGATCCGGTGACCGTGGTCGACACCGACACGGAGCGGCTCTTGCGTGATCGG TTGGCTCAACTTCGGCCCGGTGACCCGATTCTCGGGGAGGAAGGTGGTGGTCCCGCCGACGTGACGGCTA CACCCTCCGACCGGGTCACTTGGGTGCTCGACCCCATCGACGGCACGGTGAATTTCGTCTACGGCATCCC GGCGTACGCGGTGTCGATTGGGGCACAGGTTGGCGGCATCACGGTGGCGGGCGCGGTCGCCGACGTCGCC GCTCGCACGGTGTATTCGGCGGCGACGGGCCTCGGCGCACATCTCACCGATGAGCGGGGGAGACATGTGT TGCGGTGCACCGGTGTCGACGAGTTGTCGATGGCGTTGCTGGGTACCGGCTTCGGGTACTCGGTTCGGTG CCGCGAGAAGCAGGCAGAATTGCTGGCTCATGTTGTGCCGTTGGTCCGCGACGTGCGTCGGATCGGTTCT GCGGCGCTGGACTTGTGCATGGTAGCGGCGGGTCGGCTGGACGCCTACTACGAGCACGGGGTGCAGGTGT GGGACTGTGCGGCAGGTGCGTTGATCGCCGCTGAGGCGGGGGCCCGCGTGTTGTTGTCAACGCCGAGAGC GGGCGGCGCGGGGTTGGTGGTGGTGGCTGCCGCGCCCGGAATCGCCGACGAGCTGTTGGCGGCGCTACAG CGATTCAACGGCCTAGAGCCGATCCCGGACTGA
*GC% Content for gene: 68.270%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): Not available
*GC% Content for gene (codon optimized): Not available
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
*NCBI Gene #: 887210
*Protein ID (NP or XP #) or Wolbachia#: NP_217217.1
*Organism (including strain): Mycobacterium tuberculosis H37Rv
Etiologic Risk Group (see link below):
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Tuberculosis is caused by the Mycobacterium tuberculosis. It is an airborne disease that mainly attacks the respiratory system. Most cases of tuberculosis are latent and do no exhibit any symptoms. However, about 10% of latent cases will develop into active tuberculosis. Roughly 50% of cases of active tuberculosis are fatal. Roughly one-third of the global population have had tuberculosis. Infection is primarily in developing countries. Over 1 million people die from tuberculosis each year. Tuberculosis is diagnosed by a tuberculosis skin test, but it is difficult to treat the disease. Symptoms of active TB include a coughing that last longer than 3 weeks, chest pain, coughing up blood, weakness, weight less, loss of appetite, chills, fever, night sweats.
Link to TDR Targets page (if present): None present
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
https://www.patricbrc.org/portal/portal/patric/Feature?cType=feature&cId=PATRIC.1773.197.CCJS01000077.CDS.32303.33136.rev
Essentiality of this protein:
"myo-inositol is a key metabolite for mycobacteria"
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1222319/)
"Phosphatidylinositol is an essential component of mycobacteria, and phosphatidylinositolbased lipids such as phosphatidylinositolmannosides, lipomannan, and lipoarabinomannan are major immunomodulatory components of the Mycobacterium tuberculosis cell wall. Inositol monophosphatase (EC 3.1.3.25) is a crucial enzyme in the biosynthesis of free myo-inositol from inositol-1-phosphate, a key substrate for the phosphatidylinositol synthase in mycobacteria."
(http://pubs.acs.org/doi/pdf/10.1021/bi0160056)
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Multimer, 3 chains
Complex of proteins?: Trimer, Chains A B C
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1222319/pdf/11772411.pdf
*EC#: 3.1.3.25
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=3.1.3.25&Suchword=&organism%5B%5D=Mycobacterium+tuberculosis&show_tm=0
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Inositol Monophosphatase + Mg2+; spectrophotometric
Assay Method:
"IMP activity was determined by quantifying the inorganic phosphate released from substrate using the Malachite Green procedure modified according to Baykov et al. (19). Standard assays were conducted in 100-µL reaction mixture aliquots including 50 mM Tris-HCl pH 8.5, 0.4 mM I-1-P, 10 mM Mg2+ and 1 µg of the purified protein. The mixture was incubated at 37 °C for 1 min. The reaction was stopped by the addition of 700 µL of water and 200 µL of the malachite green reagent. The reagent contains 2.4 M H2SO4, which is sufficient to stop the reaction. The release of inorganic phosphate (Pi) was determined by measuring the OD at 630 nm in comparison to that of standard Pi samples. Control assays included substrate and Mg2+ but no enzyme or enzyme alone. For the determination of the Mg2+-dependent enzyme profile, pH optimum, Km, metal ion dependence, monovalent cation inhibition, substrate specificity, and temperature dependence, the corresponding factors were varied. For the determination of thermal stability, the enzyme was incubated for different periods of time at 70 °C in the presence or absence of 10 mM MgCl2. The activity was then measured by incubating the enzyme for 1 min at 37 °C in the presence of the substrate and 10 mM MgCl2."
(http://pubs.acs.org/doi/pdf/10.1021/bi0160056)
-- links to assay reagents (substrates) pages.
1mg d-myo-inositol-phosphate
https://www.caymanchem.com/app/template/Product.vm/catalog/10007777
Structure (PDB or Homology model)
PDB #: 2Q74
Current Inhibitors: None present
Expression Information (has it been expressed in bacterial cells):
E. Coli C41 (DE3)
pET23b-suhB plasmid
Method:
"An overnight culture of E. coli C41 (DE3) pET23b-suhB was used to inoculate a large volume of LB broth supplemented with 100 µg/mL ampicillin (Sigma) and incubated at 37 °C under shaking until the optical density (OD) at 600 nm reached 0.5. The culture was then transferred to fresh terrific broth supplemented with 100 µg/mL ampicillin and induced with 0.1 mM isopropyl-â-D-thiogalactopyranoside (IPTG) (Promega) overnight at 16 °C."
(http://pubs.acs.org/doi/pdf/10.1021/bi0160056)
Purification Method:
Nickel column
"The cells [E. Coli] were harvested by centrifugation and resuspended in 50 mM MOPS buffer pH 7.9. Bacteria were disrupted by probe sonication (Soniprep 150, MSE Sanyo Gallenkamp, Crawley, Sussex, U.K.; 1-cm probe) for a total time of 2.5 min in 30-s pulses with 45-s cooling intervals between pulses. The resulting extracts were centrifuged at 27000g for 20 min at 4 °C. The supernatant was collected and applied to a Ni2+-charged His-trap column (1 mL, Amersham Pharmacia Biotech) that had been equilibrated with 125 mM phosphate buffer (pH 7.5) and 0.5 M NaCl. The column was extensively washed with 50 mM imidazole in the above buffered solution and eluted with a stepwise gradient of imidazole (50-500 mM). One milliliter fractions were collected, and the presence and purity of SuhB was detected by 15% SDS-PAGE. Fractions containing pure SuhB were dialyzed against 50 mM Tris/ HCl pH 7.5 and stored at -20 °C. Protein concentrations were determined using the BCA protein assay kit (Pierce; Rockford, IL)."
(http://pubs.acs.org/doi/pdf/10.1021/bi0160056)
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MTRPDNEPARLRSVAENLAAEAAAFVRGRRAEVFGISRAGDGDGAVRAKSSPTDPVTVVD
TDTERLLRDRLAQLRPGDPILGEEGGGPADVTATPSDRVTWVLDPIDGTVNFVYGIPAYA
VSIGAQVGGITVAGAVADVAARTVYSAATGLGAHLTDERGRHVLRCTGVDELSMALLGTG
FGYSVRCREKQAELLAHVVPLVRDVRRIGSAALDLCMVAAGRLDAYYEHGVQVWDCAAGA
LIAAEAGARVLLSTPRAGGAGLVVVAAAPGIADELLAALQRFNGLEPIPD
*length of your protein in Amino Acids: 290 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 30,027 kD
Molar Extinction coefficient of your protein at 280 nm wavelength: 20190 M-1 cm-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
GTGACACGACCTGACAACGAACCCGCGCGGCTGCGCTCTGTGGCCGAAAACCTTGCTGCCGAGGCGGCGG
CCTTCGTTCGCGGTCGTCGGGCCGAGGTTTTCGGCATCTCCAGGGCGGGCGACGGCGACGGCGCGGTGCG
CGCGAAGAGCAGCCCGACCGATCCGGTGACCGTGGTCGACACCGACACGGAGCGGCTCTTGCGTGATCGG
TTGGCTCAACTTCGGCCCGGTGACCCGATTCTCGGGGAGGAAGGTGGTGGTCCCGCCGACGTGACGGCTA
CACCCTCCGACCGGGTCACTTGGGTGCTCGACCCCATCGACGGCACGGTGAATTTCGTCTACGGCATCCC
GGCGTACGCGGTGTCGATTGGGGCACAGGTTGGCGGCATCACGGTGGCGGGCGCGGTCGCCGACGTCGCC
GCTCGCACGGTGTATTCGGCGGCGACGGGCCTCGGCGCACATCTCACCGATGAGCGGGGGAGACATGTGT
TGCGGTGCACCGGTGTCGACGAGTTGTCGATGGCGTTGCTGGGTACCGGCTTCGGGTACTCGGTTCGGTG
CCGCGAGAAGCAGGCAGAATTGCTGGCTCATGTTGTGCCGTTGGTCCGCGACGTGCGTCGGATCGGTTCT
GCGGCGCTGGACTTGTGCATGGTAGCGGCGGGTCGGCTGGACGCCTACTACGAGCACGGGGTGCAGGTGT
GGGACTGTGCGGCAGGTGCGTTGATCGCCGCTGAGGCGGGGGCCCGCGTGTTGTTGTCAACGCCGAGAGC
GGGCGGCGCGGGGTTGGTGGTGGTGGCTGCCGCGCCCGGAATCGCCGACGAGCTGTTGGCGGCGCTACAG
CGATTCAACGGCCTAGAGCCGATCCCGGACTGA
*GC% Content for gene: 68.270%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): Not available
*GC% Content for gene (codon optimized): Not available
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**